Bacterial cell complex composition and method of use

ABSTRACT

The present invention relates to a composition and method useful for stimulating the immune system and for inhibiting proliferation of and inducing apoptosis in responsive cells of an animal. The present invention further relates to a composition comprising a  Mycobacterium phlei  deoxyribonucleic acid (M-DNA)- Mycobacterium phlei  cell wall complex (MCC), wherein the  Mycobacterium phlei -DNA is preserved and complexed on the  Mycobacterium phlei  cell wall, and a pharmaceutically acceptable carrier. MCC stimulates responsive cells of the immune system to produce cytokines and reactive oxygen species and inhibits proliferation of and induces apoptosis in responsive cells, including cancer cells, in an animal. Methods of making MCC and methods of using MCC also are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/054,777, filed Aug. 5, 1997 and U.S. Provisional Application Ser.No. 60/075,111, filed Feb. 18, 1998, and U.S. Provisional Application60/075,067, filed Feb. 18, 1998, and U.S. Provisional Application Ser.No. 60/086,317, filed May 21, 1998.

FIELD OF THE INVENTION

The present invention is useful for inducing a response in responsivecells of the immune system, and for inhibiting proliferation of andinducing apoptosis in cancer cells. The present invention relates to acomposition comprising a bacterial deoxyribonucleic acid (DNA)-bacterialcell wall complex, wherein the bacterial DNA is preserved and complexedon the bacterial cell wall, such that it is effective in inducing aresponse in responsive cells of an animal. More particularly, thepresent invention relates to a Mycobacterium phlei deoxyribonucleic acid(M-DNA)-Mycobacterium phlei cell wall complex (MCC), wherein the M-DNAis preserved and complexed on the Mycobacterium phlei cell wall, suchthat the MCC is effective in inducing a response in responsive cells ofan animal. Methods of making MCC and methods of using MCC are alsodisclosed.

BACKGROUND OF THE INVENTION

Preparations of biological and chemical origin including, but notlimited to, preparations from bacteria, viruses, yeast, and plants havebeen used to stimulate or to inhibit responsive cells in an animal,including a human. Among bacteria, preparations of cell wall from, butnot limited to, Mycobacterium species have been used to treat diseasesincluding, but not limited to, skin diseases U.S. Pat. No. 4,340,586),bacterial infections (U.S. Pat. No. 3,172,815), viral infections (U.S.Pat. No. 4,744,984) and cancers (U.S. Pat. No. 4,503,048). Mycobacterialcell wall extracts are composed primarily of peptidoglycan andglycolipid (Chin et al. Journal of Urology 156:1189-1193, 1996) andcontain N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) andmycolic acid derivatives. Both muramyl dipeptide and mycolic acidderivatives stimulate the immune system by activation of macrophage andmonocyte mediated reactions (Mallick et al. Comparative Immunology andMicrobiology of Infectious Diseases 8:55-63, 1985; Teware et al.Veterinary Parasitology 62:223-230, 1996). As used herein, the immunesystem is defined to include macrophages, monocytes, lymphocytes andleukocytes. These reactions, mediated by the immune system, inducecytolysis, which is the complete or partial destruction of a cell.However, the therapeutic benefits obtained using such cell wall extractsto treat cancer cells are variable and inconsistent, and appear todepend on the method by which the preparation is prepared and delivered,and on the stability of the preparation.

Activated macrophages and monocytes produce bioactive molecules thatinitiate, accelerate, amplify and modulate responsive cells of theimmune system. By produce is meant synthesize and release. Thesebioactive molecules include, but are not limited to, cytokines andreactive oxygen species. Cytokines include, but are not limited to,interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-12 (IL-12) and GM-CSF. IL-12 alone, and in combination withother cytokines, promotes the maturation of leukocytes including, butnot limited to, B-lymphocytes, CD4+ T-cells, CD8+ T-cells, and NK cellsand induces the secretion of interferon-gamma. The cytokine IL-12 isreported to have anti-cancer activity is some cancer cells lines (Stineet al. Annals NY Academy of Science 795:420-421, 1996; Chen et al.Journal of Immunology 159:351-359, 1997). This anti-cancer activityincludes, but is not limited to, activation of specific cytolyticT-lymphocytes, activation of natural killer (NK) cells and induction ofthe anti-angiogenic proteins IP-10 and MiG. IP-10 inhibits cancer growthand metastasis, inhibits cancer-induced neovascularization, and furtheractivates NK cells (Angillo et al. Annals NY Academy of Sciences795:158-165, 1996). The cytokine GM-CSF is reported to have pro-canceractivity is some cancer cells lines (Hawkyard et al. Journal of Urology150:514-518, 1993).

Reactive oxygen species include, but are not limited to, nitric oxide,superoxide radicals, and hydroxyl radicals. Nitric oxide, superoxideradicals, and hydroxyl radicals, among other activities, inducecytolysis and apoptosis in susceptible target cells.

Apoptosis is a genetically programmed, non-inflammatory,energy-dependent form of cell death in tissues including, but notlimited to, adult tissues (Steller, H. Science 267:1445-1449, 1995).Apoptosis can be initiated by ligands which bind to cell surfacereceptors including, but not limited to, Fas (CD95) (French et al.Journal of Cell Biology 133:335-343, 1996) and tumor necrosis factorreceptor 1 (TNFR1). FasL binding to Fas and TNF binding to TNFR1initiate intracellular signaling resulting in the activation of cysteineaspartyl proteases (caspases) that initiate the lethal proteolyticcascade of apoptosis execution (Muzio et al. Cell 85:817-827, 1996),which is associated with nuclear DNA-fragmentation, release of nuclearmatrix proteins (NuMA) and loss of cell substrate contact.

Apoptosis also can be induced by intracellular proteins including, butnot limited to, p53/p21. p53/p21 act as a transcription factors toinduce expression of apoptosis-mediating genes, including, but notlimited to, genes encoding proteins that generate free radicals that, inturn, damage the cell's mitochondria, whose contents leak out into thecytoplasm and activate apoptotic caspases (Polyak et al. Nature389:300-305, 1997).

Cancer is an aberrant net accumulation of atypical cells, which resultsfrom an excess of proliferation, an insufficiency of apoptosis, or acombination of the two. Mutations in apoptosis-related genes such as,but not limited to, Fas, TNFR1 and p53/p21 each have been implicated inthe pathogenesis of cancers (Levine, A. Cell 88:323-331, 1997; Fisher,D. Cell 78:529-542, 1994). Apoptosis is important not only to thepathogenesis of cancers, but also to their likelihood of resistance toanti-cancer therapies.

Resistance to apoptosis induction has emerged as an important categoryof multiple drug resistance (MDR), one that likely explains asignificant proportion of treatment failures. MDR, the simultaneousresistance to structurally and functionally unrelated chemotherapeuticagents, can be both inherent and acquired. That is, some cancers neverrespond to therapy, whereas other cancers, initially sensitive totherapy, develop drug resistance. As chemotherapeutic agents relyprimarily on induction of apoptosis in cancer cells for theirtherapeutic effect, drug resistance, which diminishes the effectivenessof chemotherapeutic agents, leads directly or indirectly to reducedapoptosis and is generally associated with poor prognosis in a varietyof cancers.

Prior art anti-cancer agents have proven to be less than adequate forclinical applications. Many of these agents are inefficient (Bischoff etal. Science 274:373-376, 1996) or toxic, have significant side effects(Lamm et al. Journal of Urology 153:1444-1450, 1995), result indevelopment of drug resistance or immunosensitization, and aredebilitating for the recipient. Moreover, many of these agents depend onFas, TNFR1 or p53/p21 for their effectiveness.

Therefore, there is a need for a novel therapeutic agent that stimulatesresponsive cells of the immune system to produce cytokines and reactiveoxygen species and which inhibits proliferation of cancer cells andinduces apoptosis in cancer cells. This therapeutic agent should beuseful as an anti-cancer agent and as an adjunct to other anti-canceragents. By adjunct is meant useful with other anti-cancer agents toincrease treatment effectiveness. Moreover, such a therapeutic agentshould be simple and relatively inexpensive to prepare, its activityshould be reproducible among preparations, its activity should remainstable over time, and its effects on cancer cells should be achievablewith dose regimens that are associated with minimal toxicity.

SUMMARY OF THE INVENTION

The present invention satisfies the above need by providing atherapeutic composition comprising a bacterial deoxyribonucleic acid(DNA)-bacterial cell wall complex, wherein the bacterial DNA ispreserved and complexed on the bacterial cell wall, such that it iseffective in inducing a response in responsive cells of an animal. Moreparticularly, the present invention provides a therapeutic compositioncomprising a mycobacterial deoxyribonucleic acid (B-DNA)-mycobacterialcell wall complex (BCC), wherein the B-DNA is preserved and complexed onthe mycobacterial cell wall, such that the BCC is effective in inducinga response in responsive cells of an animal. Most particularly, thepresent invention relates to a Mycobacterium phlei deoxyribonucleic acid(M-DNA)-Mycobacterium phlei cell wall complex (MCC), wherein the M-DNAis preserved and complexed on the Mycobacterium phlei cell wall, suchthat the MCC is effective in inducing responsive cells of the immunesystem to produce cytokines and reactive oxygen species, and ininhibiting proliferation of and inducing apoptosis in responsive cellsincluding, but not limited to, cancer cells, in an animal.

MCC is simple and relatively inexpensive to prepare; its activity isreproducible among preparations; it remains stable and effective overtime; and, it is effective at dose regimens that are associated withminimal toxicity.

To prepare MCC, the Mycobacterium phlei (M. phlei) are grown in liquidmedium and harvested. The M. phlei are disrupted and the solidcomponents of the disrupted M. phlei are collected by centrifugalsedimentation. The solid components are deproteinized, delipidated, andwashed. DNase-free reagents are used to minimize M-DNA degradationduring preparation.

MCC is effective as a therapeutic agent in preventing, treating andeliminating a variety of diseases including, but not limited to,malignant, autoimmune and immunodeficiency diseases. It is particularlyuseful for treating diseases and processes mediated by undesired anduncontrolled cell proliferation, such as cancers. MCC also is effectiveas an adjunct to enhance the effectiveness of other anti-cancer agents.Such agents include, but are not limited to, drugs, immunostimulants,antigens, antibodies, vaccines, radiation and chemotherapeutic, genetic,biologically engineered and chemically synthesized agents, and agentsthat target cell death molecules for activation or inactivation, thatinhibit proliferation of, and that induce apoptosis in cancer cells.

MCC, in a pharmaceutically acceptable carrier, is administered to ananimal in a dosage effective to stimulate a response in responsive cellsof the immune system, and to inhibit proliferation of and induceapoptosis in responsive cells. MCC can be administered by methodsincluding, but not limited to, suspension in aqueous formulations,emulsification in oil or other hydrophobic liquid formulations,enclosure in liposomes, and complexion with natural or artificialcarriers, with tissue- or cell-specific ligands or with tissue- orcell-specific antibodies.

Accordingly it is an object of the present invention to provide acomposition and method that induces a therapeutic response in responsivecells of an animal, including a human.

Another object of the present invention to provide a composition andmethod that stimulates responsive cells of the immune system.

Another object of the present invention is to provide a composition andmethod that stimulates responsive cell of the immune system to producebioactive molecules.

Another object of the present invention is to provide a composition andmethod that stimulates responsive cells of the immune system to producecytokines.

Another object of the present invention is to provide a composition andmethod that stimulates responsive cells of the immune system to produceIL-10.

Another object of the present invention is to provide a composition andmethod that stimulates responsive cells of the immune system to produceIL-12.

Another object of the present invention is to provide a composition andmethod that stimulates responsive cells of the immune system to producereactive oxygen species.

Another object of the present invention is to provide a composition andmethod that is effective for treating a disease in an animal.

Another object of the present invention is to provide a composition andmethod that is effective to prevent a cancer in an animal.

Another object of the present invention is to provide a composition andmethod that is effective to treat a cancer in an animal.

Another object of the present invention is to provide a composition andmethod that is effective to eliminate a cancer in an animal.

Another object of the present invention is to provide a composition andmethod that inhibits proliferation of responsive cells of an animal.

Another object of the present invention to provide a composition andmethod that induces apoptosis in responsive cells of an animal.

Another object of the present invention is to provide a composition andmethod that induces apoptosis independent of Fas.

Another object of the present invention is to provide a composition andmethod that induces apoptosis independent of TNFR1.

Another object of the present invention is to provide a composition andmethod that induces apoptosis independent of p53/p21.

Another object of the present invention is to provide a composition andmethod that induces apoptosis independent of drug resistance.

Another object of the present invention is to provide a composition andmethod that activates caspases in responsive cells of an animal.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to other anti-cancer therapies.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to chemical agents.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to radiation therapy.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to biological agents.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to biologically engineeredagents.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to vaccines.

Another object of the present invention is to provide a composition andmethod that is effective as an adjunct to nucleic acid-based vaccines.

Another object of the present invention is to provide a composition andmethod that is effective in inhibiting angiogenesis in a cancer.

Another object of the present invention is to provide a composition andmethod that is effective in restoring the immune system in animmunosuppressed animal regardless of the cause of theimmunosuppression.

Another object of the present invention is to provide a composition andmethod that is effective in treating an immunodeficiency disease.

Another object of the present invention is to provide a composition andmethod that induces terminal differentiation of incompletelydifferentiated cells.

Another object of the present invention is to provide a composition ofparticle size and formulation that is optimal for recognition byresponsive cells.

Another object of the present invention is to provide a composition ofparticle size and formulation that is optimal for interaction withresponsive cells.

Another object of the present invention is to provide a composition ofparticle size and formulation that is optimal for uptake by responsivecells.

Another object of the present invention is to provide a composition thatcan be prepared in large amounts.

Another object of the present invention is to provide a composition thatis relatively inexpensive to prepare.

Another object of the present invention is to provide a composition thathas reproducible activity among preparations.

Another object of the present invention is to provide a composition thatremains stable over time.

Another object of the present invention is to provide a composition thatmaintains its effectiveness over time.

Another object of the present invention is to provide a composition thatis minimally toxic to the recipient.

Another object of the present invention is to provide a composition thatwill not cause anaphylaxis in the recipient.

Another object of the present invention is to provide a composition thatdoes not sensitize the recipient to tuberculin skin tests.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. MCC stimulation of IL-6, IL-12 and GM-CSF production in humanHT-1197 and HT-1376 bladder cancer cells, human THP-1 monocytes, murinemacrophages, murine RAW 264.7 monocytes and murine spleen cells by MCC.Results are the mean±SD of 3 independent experiments.

FIG. 2. MCC and MCC-DNA stimulation of IL-12 production in human THP-1monocytes in the absence and in the presence of CD14 antibodies.

FIG. 3. MCC, M. phlei-DNA and MCC-DNA stimulation of IL-12 production inhuman THP-1 monocytes in the absence and in the presence of cytochalasinD.

FIG. 4. MCC, MCC-DNA and REGRESSIN® stimulation of IL-12 production inmurine macrophages before and after DNase I treatment.

FIG. 5. Stimulation of IL-12 production in murine macrophages by PBS,interferon-gamma and MCC and by interferon gamma+MCC.

FIG. 6. MCC stimulation of NO production in RAW 264.7 monocytes.

FIGS. 7A. and 7B. In vivo stimulation of cytokine production in CD-1mice by intraperitoneal administration of MCC (7A) and intravenousadministration of MCC (7B).

FIG. 8. MCC inhibition of proliferation of HT-1376, HT-1197, B-16 F1,THP-1, RAW 264.7, Jurkat, HL-60 and HL-60 MX-1 cancer cells. Results arethe mean±SD of 3 independent experiments

FIGS. 9A. and 9B. Inhibition of proliferation of HT-1376. HT-1197, B-16F1, THP-1, RAW 264.7, Jurkat, HL-60, HL-60 MX-1 cancer cells by M.phlei-DNA (9A), MCC-DNA (9B), calf thymus-DNA (9A & 9B) and herringsperm-DNA (9A & 9B). Results are the mean±SD of 3 independentexperiments.

FIG. 10. Inhibition of proliferation of human leukemic THP-1 monocytesby MCC, M. phlei-DNA, MCC-DNA and hIL-12. Results are the mean±SD of 3independent experiments.

FIGS. 11A. and 11B. Inhibition of proliferation of HT-1197 (11A) andHT-1376 (11B) human bladder cancer cells by MCC and LPS. Results are themean±SD of 3 independent experiments.

FIG. 12. Induction of DNA fragmentation in human leukemic THP-1monocytes by PBS, MCC, DNase I treated MCC, M. phlei-DNA, DNase Itreated M. phlei-DNA, and herring sperm-DNA. Results shown are for 1 of3 experiments, each of which gave similar results.

FIGS. 13A. and 13B. Induction of DNA fragmentation in HT-1197 (13A) andHT-1376 (13B) human bladder cancer cells by MCC, and hIL-12. Resultsshown are for 1 of 3 experiments, each of which gave similar results

FIG. 14. Release of NuMA from human leukemic THP-1 monocytes by MCC, M.phlei-DNA, MCC-DNA and herring sperm-DNA. Results are the mean±SD of 3independent experiments.

FIG. 15. Release of NuMA from human leukemic THP-1 monocytes byuntreated and DNase I treated MCC, M. phlei-DNA and MCC-DNA. Results arethe mean±SD of 3 independent experiments.

FIG. 16. Release of NuMA from HT-1197 and HT-1376 human bladder cancercells with increasing concentrations of MCC. Results are the mean±SD of3 independent experiments.

FIGS. 17A. and 17B. Release of NuMA from HT-1197 (17A) and HT-1376 (17B)human bladder cancer cells with 1 μg/ml MCC or with 100 μg/ml MCC over48 h. Results are the mean±SD of 3 independent experiments.

FIG. 18. NuMA release from Jurkat cells incubated with PBS, CH-11antibodies, ZB4 antibodies, M. phlei-DNA, CH-11 antibodies+M. phlei-DNAand ZB4 antibodies+M. phlei-DNA.

FIG. 19. Effect of BstU I restriction endonuclease on nuclearfragmentation of M. phlei-DNA and of methylated M. phlei-DNA.

FIG. 20. NuMA release from human leukemic THP-1 monocytes by M.phlei-DNA and by sonicated, BstU I cleaved, autoclaved, and methylatedM. phlei-DNA.

FIG. 21. In vivo anti-cancer activity of MCC and of DNase I treated MCCin line 10 hepatoma in guinea pigs. Results are the mean±SD of 7 animalsin each experimental group.

FIG. 22. Percentage LDH release by HT-1197 and HT-1376 human bladdercancer cells as an indicator of MCC cytotoxicity. Results are themean±SD of 3 independent experiments.

FIG. 23. Stability of MCC during 6 months of storage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition comprising a bacterialdeoxyribonucleic acid (DNA)-bacterial cell wall complex, wherein thebacterial DNA is preserved and complexed on the bacterial cell wall,such that it is effective in inducing a response in responsive cells ofan animal. More particularly, the present invention relates to acomposition comprising a mycobacterial deoxyribonucleic acid(B-DNA)-mycobacterial cell wall complex (BCC), wherein the B-DNA ispreserved and complexed on the mycobacterial cell wall, such that theBCC is effective in inducing a response in responsive cells of ananimal. Most particularly, the present invention relates to a M. phleideoxyribonucleic acid (M-DNA)-M. phlei cell wall complex (MCC) and apharmaceutically acceptable carrier, wherein the M-DNA is preserved andcomplexed on the M. phlei cell wall. MCC is effective in inducingresponsive cells of the immune system to produce cytokines and reactiveoxygen species, and in inhibiting proliferation of and inducingapoptosis in responsive cells including, but not limited, to cancercells, in an animal. In MCC, the amount of M-DNA is enriched relative tothe amount of M-DNA in an intact M. phlei cell. Further, the M-DNA ispreserved and complexed on the M. phlei cell wall so that it is moreaccessible to the responding cells than is the M-DNA within an intact M.phlei cell.

Many bacterial species can be used to practice the present inventionincluding, but not limited to, Coryneform species, Corynebacteriumspecies, Rhodococcus species, Eubacterium species, Bordetella species,Escherichia species, Listeria species, Nocardia species andMycobacterium species. Preferably, Mycobacterium species including, butnot limited to, M. smegmatis, M. fortuitum, M. kansaii, M. tuberculosis,M. bovis, M. vaccae, M. avium and M. phlei are used. More preferably,the Mycobacterium species M. phlei is used.

MCC is simple and relatively inexpensive to prepare, its activity isreproducible among preparations and remains stable over time. Further,it is minimally, if at all, toxic to the recipient, does not cause apositive tuberculin reaction in the recipient and rarely causes ananaphylactic response in the recipient even upon repeatedadministration.

Although not wanting to be bound by the following hypothesis, it isbelieved that the oligonucleotide sequences and structures of M-DNA arenecessary for biological activity of MCC, and that the complexion ofM-DNA on the deproteinized, delipidated M. phlei cell wall is importantfor optimal biological activity of MCC. It is to be understood thatM-DNA alone, or M-DNA complexed on a carrier other than M. phlei cellwall, can be used to induce a response in responsive cells of an animal.That is, methods disclosed for using MCC can also be used for M-DNA.

The M-DNA content of MCC preferably is between about 0.001 mg/100 mg dryMCC and about 90 mg/100 mg dry MCC, more preferably between about 0.01mg/100 mg dry MCC and about 40 mg/100 mg dry MCC, most preferablybetween about 0.1 mg/100 mg dry MCC and about 30 mg/100 mg dry MCC.Also, it is preferable that the protein content be less than about 2mg/100 mg dry MCC and that the fatty acid content be less than about 2mg/100 mg dry MCC.

Methods to increase the therapeutic activity of MCC include, but are notlimited to, chemically supplementing or biotechnologically amplifyingstimulatory sequences or confirmations of DNA derived from the same ordifferent bacterial species, or using bacterial plasmids containingappropriate stimulatory sequences or confirmations of DNA derived fromthe same or different bacterial species. Other methods to increase thetherapeutic activity of MCC include, but are not limited to, complexingthe MCC to natural or synthetic carriers or coupling the MCC totissue-type or cell-type directed ligands or antibodies.

Administration of MCC is not an immunization process. It is atherapeutic treatment that stimulates a response in responsive cells ofthe immune system, and that inhibits proliferation of and inducesapoptosis in responsive cells. This therapeutic treatment is useful toprevent, treat or eliminate a disease including, but not limited to, acancer. Moreover, the unexpected and surprising ability of MCC to induceapoptosis in various cancer cells including, but not limited to, Fasabnormal, p52/21 abnormal and drug resistant cancer cells, addresses along felt unfulfilled need in the medical arts, and provides animportant benefit for animals, including humans.

Although not wanting to be bound by the following hypothesis, it isbelieved that the therapeutic effects of MCC include, but are notlimited to, stimulation of responsive cells of the immune system toproduce cytokines and reactive oxygen species, which cause cytolysis andapoptosis in responsive cells, and induction of caspase activity inresponsive cells, which causes apoptosis. Cytolysis and apoptosis, bothindividually and in combination, have both anti-cancer activity andadjunct activity. That is, MCC can be used alone as an anti-cancer agentand MCC can be used before, at the same time as, or after anotheranti-cancer agent to increase treatment effectiveness.

MCC and its pharmaceutically acceptable carrier may be prepared byvarious techniques. Such techniques include bringing into associationthe MCC and its carriers or excipients. Preferably, the MCC compositionsare prepared by uniformly and intimately bringing into association theMCC with liquid carriers, with solid carriers, or with both. Liquidcarriers include, but are not limited to, aqueous formulations,non-aqueous formulations, or both. Solid carriers include, but are notlimited to, biological carriers, chemical carriers, or both.

MCC may be administered in an aqueous suspension, an oil emulsion, waterin oil emulsion and water-in-oil-in-water emulsion, and in carriersincluding, but not limited to, liposomes, microparticles, site-specificemulsions, long-residence emulsions, sticky-emulsions, microemulsions,nanoemulsions, microspheres, nanospheres, nanoparticles and minipumps,and with various natural or synthetic polymers that allow for sustainedrelease of MCC, the minipumps or polymers being implanted in thevicinity of where drug delivery is required. Polymers and their use aredescribed in, for example, Brem et al. (Journal of Neurosurgery74:441-446, 1991). Further, MCC can be used with any one, all, or anycombination of excipients regardless of the carrier used to present theMCC to the responding cells. These include, but are not limited to,anti-oxidants, buffers, and bacteriostats, and may include suspendingagents and thickening agents.

Preferably, MCC is administered as an aqueous suspension. Foradministration in an aqueous carrier, MCC is suspended in apharmaceutically acceptable buffer including, but not limited to, salineand phosphate buffered saline (PBS) by techniques including, but notlimited to, sonication and microfluidization, and is either ascepticallyprocessed or terminally sterilized. For example, freeze-dried(lyophilized) MCC may be stored in sealed ampoules or vials requiringonly the addition of a carrier, for example sterile water, immediatelyprior to use.

For administration in a non-aqueous carrier, MCC is emulsified with amineral oil or with a neutral oil such as, but not limited to, adiglyceride, a triglyceride, a phospholipid, a lipid, an oil andmixtures thereof, wherein the oil contains an appropriate mix ofpolyunsaturated and saturated fatty acids. Examples include, but are notlimited to, soybean oil, canola oil, palm oil, olive oil and myglyol,wherein the number of fatty acid carbons is between 12 and 22 andwherein the fatty acids can be saturated or unsaturated. Optionally,charged lipid or phospholipid can be suspended in the neutral oil.

The size of the MCC particles administered should be optimal forrecognition of, interaction with, and uptake by the responsive cells.Preferably, the mean diameter of the MCC particles is between about 10and about 10,000 nm, more preferably between about 100 and about 1000 nmand most preferably between about 250 and about 600 nm.

MCC is administered in an amount effective to induce a therapeuticresponse in an animal, including a human. The dosage of MCC administeredwill depend on the condition being treated, the particular formulation,and other clinical factors such as weight and condition of the recipientand route of administration. Preferably, the amount of MCC administeredis from about 0.00001 mg/kg to about 100 mg/kg per dose, more preferablyfrom about 0.0001 mg/kg to about 50 mg/kg per dose, and most preferablyfrom about 0.001 mg/kg to about 10 mg/kg per dose.

M-DNA also is administered in an amount effective to induce atherapeutic response in an animal, including a human. The dosage ofM-DNA to be administered will depend on the condition being treated, theparticular formulation, and other clinical factors such as weight andcondition of the recipient and route of administration. Preferably theamount of M-DNA administered is from about 0.00001 mg/kg to about 100mg/kg per dose, more preferably from about 0.0001 mg/kg to about 50mg/kg per dose and most preferably from about 0.001 mg/kg to about 10mg/kg per dose.

Routes of administration include, but are not limited to, oral, topical,subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal,intrathecal, intralesional, intratumoral, intrabladder, intra-vaginal,intraocular, intrarectal, intrapulmonary, intraspinal, transdermal,subdermal, placement within cavities of the body, nasal inhalation,pulmonary inhalation, impression into skin and electrocorporation.

Depending on the route of administration, the volume per dose ispreferably about 0.001 ml to about 100 ml, more preferably about 0.01 mlto about 50 ml, and most preferably about 0.1 ml to about 30 ml. MCC canbe administered in a single dose treatment or in multiple dosetreatments on a schedule and over a period of time appropriate to thedisease being treated, the condition of the recipient and the route ofadministration.

MCC is effective as therapeutic agent for preventing, treating oreliminating a disease including, but not limited, to a cancer. Suchcancers include, but are not limited to, squamous cell carcinoma,fibrosarcoma, sarcoid carcinoma, melanoma, mammary cancer, lung cancer,colorectal cancer, renal cancer, osteosarcoma, cutaneous melanoma, basalcell carcinoma, pancreatic cancer, bladder cancer, ovarian cancer,leukemia, lymphoma and metastases derived therefrom.

MCC retains its therapeutic effectiveness after sonication andautoclaving, which reduce oligonucleotide length, and after CpGmethylation, which abolishes activity of the palindromic oligonucleotidesequence purine-purine-C-G-pyrimidine-pyrimidine. MCC does not retainits therapeutic effectiveness after DNase I treatment, which digests theM-DNA.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1 Preparation of MCC from Mycobacterium phlei

MCC was prepared from Mycobacterium phlei (strain 110). M. phlei wasobtained from the Institut fur Experimental Biologie and Medizin,Borstel, Germany, and was stored as a suspension in sterile milk at −60°C. The M. phlei was cultured on Petragnani medium (Difco Labs, Detroit,Mich.) and was grown in granulated agar BACTO® AC broth (Difco Labs) for10 to 20 days. The cells were harvested by centrifugal sedimentation.All reagents used in the following procedure were selected to enhanceconservation of the M. phlei DNA.

About 400 grams of moist cell mass was placed into an autoclaved blenderwith a capacity of about 1200 ml. The cell mass was mixed at high speedfor between 30 to 60 sec. After mixing, 6 ml of DNase-freepolyoxgenthylenesorbitan monooleate, Tween 80, (Sigma Chemical Co., St.Louis, Mo.) and between 200 and 400 ml of autoclaved water were added tothe cell mixture. The entire cell suspension was again mixed in theblender at low speed for about 10 sec.

Cell disruption was accomplished by sonication. Five hundred ml of cellsuspension, wherein the cells comprised about 50% to 70% of the volume,were placed in a one liter autoclaved beaker and sonicated. The sonicatewas stored in an autoclaved flask on ice during the fractionationprocess. Unbroken cells were remove by low speed centrifugation. Thesupernatant from the low speed centrifugation was centrifuged for 1 h at27,500 g at 15° C. and the supernatant from this centrifugation wasdiscarded.

The sediment from the 27,000 g centrifugation was transferred to anautoclaved blender and suspended in autoclaved deionized water by mixingat low speed. This suspension was again centrifuged at 27,000 g at 15°C. for 1 h and the supernatant was again discarded. The sediment wassuspended in autoclaved deionized water and spun for 5 min at 350 g tosediment any remaining unbroken cells. The supernatant was decanted andcentrifuged at 27,000 g for 1 h at 15° C. to sediment the crude cellwall fraction.

The crude cell wall fraction was deproteinized by digestion withproteolytic enzymes, care being taken to use DNase-free reagents wherepossible to optimize the amount of M-DNA in the preparation and topreserve the structure of the M-DNA in the preparation. The crude cellwall fraction derived from about 400 g of whole cells was suspended in 1liter of 0.05 M DNase-free Tris-HCl, pH 7.5, by mixing at low speed.After the crude cell wall fraction was thoroughly suspended, 50 mg ofDNase-free trypsin (Sigma Chemical Co) was added and the suspension wasstirred using a magnetic stirring bar at 35° C. for 6 h. Followingtrypsin treatment, 50 mg of DNase-free pronase (Amersham Canada Limited,Oakville, Ontario) were added to each liter of trypsin-treated crudecell wall suspension. The suspension was stirred using a magneticstirring bar for 12 to 18 h at 35° C.

After proteolytic digestion, the crude cell wall fraction wasdelipidated with detergent and phenol. To each liter of suspension, 60 gof DNase-free urea (Sigma Chemical Co.), 2.0 ml of DNase-free 100%phenol or 150 ml of 90% w/v phenol (Sigma Chemical Co.) were added. Theflask containing the suspension was covered loosely with aluminum foil,warmed to 60-80° C. and stirred for 1 hr. The suspension was spun for 10min at 16,000 g. The supernatant fraction and the fluid beneath thepellet were discarded. The pellet was washed 3 times by resuspension inabout 1 liter of autoclaved deionized water and centrifuged for 10 minat 16,000 g.

The washed, deproteinized, delipidated MCC was lyophilized bytransferring the suspension to an autoclaved lyophilizing flask with asmall amount of autoclaved water. One 300 ml lyophilizing flask was usedfor each 30 grams of wet cell complex starting material. The MCCsuspension was shell frozen by rotating the flask in ethanol cooled withsolid carbon dioxide. After the content of the flask was frozen, theflask was attached to a lyophilization apparatus (Virtis Co. Inc.,Gardiner, N.Y.) and lyophilized. After lyophilization, the sample wastransferred to an autoclaved screw-cap container and stored at −20° C.in a desiccator jar containing anhydrous calcium sulfate.

Unless stated otherwise, the lyophilized MCC was resuspended inautoclaved deionized water or in a pharmaceutically acceptableDNase-free buffer such as, but not limited to, saline and PBS, andemulsified by sonication. Optionally, the sonicated MCC was homogenizedby microfluidization at 15,000-30,000 psi for one flow-through. The MCCsuspension was either processed under aseptic conditions or wassterilized by autoclaving.

EXAMPLE 2 Purification of M-DNA from MCC and from M. phlei

MCC was prepared as in Example 1. M-DNA was purified from MCC (MCC-DNA)by phenol/chloroform/isoamyl alcohol extraction and ethanolprecipitation (Short Protocols in Molecular Biology, 3rd Edition,Ausubel et al. Eds., John Wiley & Sons Inc., New York, USA).Unexpectedly, we found that at least about 3.6% of the dry weight of MCCis extractable M-DNA.

M-DNA was purified from M. phlei (M. phlei-DNA) by suspending the M.phlei (strain 110) in 5 ml of DNase-free 50 mM Tris-HCl, 5 mM EDTA, pH8.0, adding DNase-free lysozyme (Sigma Chemical Co.) to a concentrationof 1 mg/ml and incubating for 90 min at 37° C. DNase-free Proteinase K(Life Technologies, Burlington, Ontario, Canada) was added to aconcentration of 0.1 mg/ml, DNase-free sodium dodecyl sulfate (BioRad,Richmond, Calif.) was added to a concentration of 1% and the incubationwas continued for 10 min at 65° C. The M. phlei-DNA wasphenol/chloroform/isoamyl alcohol extracted and ethanol precipitated.

Unless stated otherwise, the M-DNA (MCC-DNA and M. phlei-DNA) wassonicated in autoclaved deionized water or in a DNase-freepharmaceutically acceptable buffer such as, but not limited to, salineand PBS. MCC, MCC-DNA and M. phlei-DNA do not contain endotoxins asdetermined using a Limulus amebocyte lysate QCL-1000 kit (BioWhittaker,Walkersville, Md.).

EXAMPLE 3 Preparation of Bacterial-DNA-bacterial Cell Wall Complex andof Bacterial DNA from other Bacterial Species

Bacterial DNA-bacterial cell wall complex is prepared from M. smegmatis,M. fortuitous, Nocardia rubra, Nocardia asteroides, Comybacteriumparvum, M. kansaii, M. tuberculosis, and M. bovis as in Example 1.Bacterial-DNA is purified from bacterial DNA-bacterial cell wall complexand from intact bacteria as in Example 2.

EXAMPLE 4 DNase Treatment

MCC-DNA, M-phlei-DNA and MCC, each containing 1 μg of M-DNA, andREGRESSIN® (U.S. Pat. No. 4,744,984) were digested with 1 internationalunit (IU) of RNase-free DNase I (Life Technologies) for 1 h at 25° C. in20 mM Tris HCl, pH 8.4, 2 mM MgCl₂ and 50 mM KCl. DNase I wasinactivated by the addition of EDTA to a final concentration of 2.5 mMand heating for 10 min at 65° C. DNase I digests both single strandedand double stranded DNA. Digestion with DNase I results in almost totaldegradation of DNA. REGRESSIN® (Bioniche, Inc. London, Ontario, Canada)is a formulation containing 1 mg mycobacterial cell wall extract, 20 μlmineral oil NF in 1 ml PBS and 0.5% v/v of TWEEN 80.

EXAMPLE 5 Cells and Reagents

All cell lines, except OC2 and SW260, were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.) and were cultured in themedium recommended by the ATCC. OC2 and SW260 were obtained from Dr. J.K. Collins (University College Cork, Cork, Ireland) and were cultured inDMEM medium supplemented with 10% FCS. Table 1 shows the cell lines,their origins and their properties.

TABLE 1 Cell lines CELL LINE ORIGIN PROPERTIES THP-1 Human acutemonocytic leukemia HL-60 Human promyelo- cytic leukemia HL-60 MX-1 Humanpromyelo- Atypical drug resist- cytic leukemia ance to mixoxantrone RAW264.7 Murine monocytic leukemia JURKAT Human T lymphoblast HT-1376 Humanbladder Mutation in p53/p21 carcinoma MDR HT-1197 Human bladdercarcinoma B-16-F1 Murine melanoma SW260 Human colon FAS-L resistanceadenocarcinoma OC2 Human esophageal carcinoma LS1034 Human cecumConventional carcinoma MDR

Murine macrophages were obtained from female CD1 mice injectedintraperitoneally with 1.5 ml sterile Brewer's thioglycolate broth(Difco, Detroit, Mich.). The peritoneal exudate (>85% macrophages) washarvested at day 4, washed by centrifugation in HBSS and cultured inRPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 20 mMHEPES (Life Technologies). The cells were allowed to adhere for 18 hafter which non-adherent cells were removed by gentle washing with warmmedium.

Murine spleen cells were prepared by gentle teasing through sterilestainless steel screens. Cell suspensions were layered on Lympholyte-Mcell separation media (CedarLane, Hornby, Ontario, Canada) andcentrifuged at 2200 rpm for 30 min to remove red blood cells and deadcells. These cells were cultured in RPMI-1640 medium supplemented with10% FCS, 2 mM L-glutamine and 20 mM HEPES (Life Technologies).

Unless stated otherwise, cells were seeded in 6 well flat-bottom tissueculture plates at concentrations between 3×10⁵ and 10⁶ cells/ml and weremaintained at 37° C. in a 5% CO2 atmosphere.

Calf thymus-DNA, herring sperm-DNA and Escherichia colilipopolysaccharide (LPS) were obtained from Sigma Chemical Co.Recombinant human IL-12 (hIL-12) was obtained from R&D Systems(Minneapolis, Minn.).

EXAMPLE 6 Stimulation of Cytokine Synthesis in vitro

The cytokine IL-12 is reported to have anti-cancer activity in somecancer cells (Voest et al. Journal National Cancer Institute 87:581-586,1995; Stine et al. Annals NY Academy of Science 795:420-421, 1996),whereas the cytokine GM-CSF is reported to have pro-cancer activity insome cancer cells (Hawkyard et al. Journal of Urology 150:514-518,1993). Moreover, some cancer cells are reported to secrete cytokines (DeReijke et al. Urology Research 21:349-352, 1993; Bevers et al. BritishJournal of Urology 80:35-39, 1997). Therefore, the effect of MCC onIL-6, IL-12 and GM-CSF synthesis by HT-1197 and HT 1376 human bladdercancer cells and by human THP-1 monocytes, murine macrophages, murineRAW 264.7 monocytes, and murine spleen cells was determined.

Cytokine production was determined in pg/ml in 100 μl of culturesupernatant using the appropriate commercial ELISA (BioSource, CamarilloCalif.). The IL-12 ELISA measures both IL-12 p70 complex and free p40subunit.

HT-1197 and HT-1376, THP-1, macrophage, RAW 264.7, and murine spleencells were incubated for 48 h with 1 μg/ml MCC. As shown in FIG. 1, MCCstimulated production of IL-6 and of IL-12 by human monocytes and murinemacrophages, but not by human bladder cancer cells, murine monocytes orspleen cells. MCC did not stimulate GM-CSF production of in any of thecancer cells tested.

These data show that MCC stimulates production of the anti-cancercytokines IL-6 and IL-12 by human monocytes and murine macrophages. MCCdoes not stimulate cytokine production by human bladder cancer cells.MCC does not stimulate production of the pro-cancer cytokine GM-CSF.

EXAMPLE 7 Effect of Untreated and DNase I Treated MCC, M. phlei-DNA,MCC-DNA and REGRESSIN® on IL-12 Production by Human THP-1 Monocytes

Human THP-1 monocytes were incubated for 48 h with MCC, M-phlei-DNA,MCC-DNA and REGRESSIN® before treatment with DNase I, after treatmentwith DNase I and after addition of M-DNA to DNase I treated MCC, M.phlei-DNA and MCC-DNA.

TABLE 2 IL-12 production in pg/ml by human THP-1 monocytes IL-12production, pg/ml culture supernatant M. phlei-DNA MCC-DNA MCCRegressin ® Treatment 1 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml None 407 676 1222196 DNase 178 367 729 200 DNase + 424 658 783 Not Done 1 μg DNA

As shown in Table 2, MCC, M. phlei-DNA and MCC-DNA each stimulated THP-1monocytes to produce IL-12. MCC stimulated more IL-12 production than M.phlei-DNA and MCC-DNA. REGRESSIN® stimulated significantly less IL-12production. DNase I treatment reduced by about 50% MCC, M. phlei-DNA andMCC-DNA stimulated IL-12 production. REGRESSIN® was not effected byDNase I treatment. Addition of MCC-DNA to DNase I-treated MCC did notrestore its IL-12 production, whereas addition of M. phlei-DNA to DNaseI-treated M. phlei and of MCC-DNA to DNase I treated MCC-DNA did restoretheir IL-12 production.

These data show that M-DNA, as MCC, M. phlei-DNA or MCC-DNA, stimulatesproduction of the cytokine IL-12 by human monocytes. MCC stimulates moreIL-12 production than M. phlei-DNA or MCC-DNA, suggesting that thecarrier on which M-DNA is presented to the monocytes can optimize theirresponse. DNase I treatment, which degrades the M-DNA, reducessignificantly M. phlei-DNA, MCC-DNA and MCC stimulation of IL-12production, suggesting that the undegraded oligonucleotide sequence ofM-DNA is necessary for stimulation of IL-12 production by humanmonocytes. Addition of M-DNA to DNase I treated MCC did not restore itsstimulation of IL-12 production, suggesting that the physical complexionof M-DNA with M. phlei cell wall in MCC is important for optimalstimulation of IL-12 production by human monocytes.

Human THP-1 monocytes were incubated for 48 h with 0.5, 1.0. 1.5 and 5.0μg/ml of MCC and of REGRESSIN®.

TABLE 3 IL-12 production in pg/ml by human THP-1 monocytes MCCRegressin ® 0.5 μg/ml  614 ± 30  65 ± 11 1.0 μg/ml 1078 ± 40 223 ± 202.5 μg/ml 1237 ± 82 313 ± 23 5.0 μg/ml  1231 ± 112 359 ± 43

As shown in Table 3, at each of the concentrations tested, MCCstimulated significantly more IL-12 production than REGRESSIN®.

EXAMPLE 8 Effect of CD14 Antibody Treatment on MCC and MCC-DNAStimulated IL-12 Production by Human THP-1 Monocytes

Human THP-1 monocytes were incubated with PBS or with 10 μg/ml ofanti-CD14 antibody (clone MY4, Coulter-Immunotech, Hialeah, Fla.) for 1h. Then, 5 μg/ml of MCC or of MCC-DNA were added and the incubation wascontinued for 48 h. CD14 antibodies, which bind to CD14 receptors on thecell surface, caused about a 20% decrease in MCC in and about an 85%decrease in MCC-DNA stimulated IL-12 production. (FIG. 2).

EXAMPLE 9 Effect of Cytochalasin D on MCC, M. phlei-DNA and MCC-DNAStimulated IL-12 Production by Human THP-1 Monocytes

Human THP-1 monocytes were incubated for 48 h with PBS or with 1 μg/mlMCC, M. phlei-DNA or MCC-DNA in the absence of and in the presence of 10μg/ml of cytochalasin D (Sigma Chemical Co.). Cytochalasin D, whichinhibits phagocytosis, caused about a 55% decrease in MCC, about a 65%decrease in M-phlei-DNA, and about a 50% decrease in MCC-DNA stimulatedIL-12 production (FIG. 3).

Although not willing to be bound by the following hypothesis, but basedon the data shown in FIGS. 2 & 3, it is believed that MCC, MCC-DNA andM. phlei-DNA interact with monocytes by more than one mechanism. FIG. 2.suggests they interact with the GPI-linked membrane receptor CD14 andare internalized. This mechanism is more specific for soluble MCC-DNAand M. phlei-DNA than for insoluble MCC. FIG. 3 suggests they interactwith phagocytic receptors, such as the scavenger receptor, and areinternalized. This mechanism is more specific for insoluble MCC than forsoluble MCC-DNA and M. phlei-DNA.

EXAMPLE 10 Effect of CG Sequence and of MCC on IL-12 Production by HumanTHP-1 Monocytes

Nucleic acid preparations from bacillus Calmette-Guerin (BCG) arereported to stimulate lymphocyte proliferation, secretion of IL-6 andIL-12 by B-lymphocytes, secretion of IL-12 by monocytes, secretion ofIL-6 and interferon-gamma by T-lymphocytes and secretion ofinterferon-gamma by NK cells (Klinman et al. Proceeding of the NationalAcademy of Science USA 93:2879-2883, 1996). The active constituent inBCG nucleic acid has been identified as the palindromic oligonucleotidesequence purine-purine-C-G-pyrimidine-pyrimidine (CG motif).

Human THP-1 monocytes were incubated for 48 h with 0.5, 1 and 5 μg/ml ofMCC or of 5′-GCTAGACGTTAGCGT-3′ DNA (SEQ ID NO 1) prepared by solidphase synthesis using an automated DNA synthesizer.

TABLE 4 Effect of CG-containing oligonucleotide and of MCC on IL-12production in pg/ml by THP-1 monocytes. 5 μg/ml 1 μg/ml 0.5 μg/mlGCTAGACGTTAGCGT Undetectable Undetectable Undetectable (SEQ ID NO 1) MCCNot Done 1239 Not Done

As shown in Table 4, the CG-containing oligonucleotide did not stimulateIL-12 production at any of the three concentrations tested, whereas MCCat 1 μg/ml had a significant stimulatory effect on IL-12 production byhuman monocytes.

EXAMPLE 11 Effect of Autoclaving on MCC and M. phlei-DNA Stimulation ofIL-12 Production by Human THP-1 Monocytes

Human THP-1 monocytes were incubated for 48 h with MCC and M. phlei-DNAand with MCC and M. phlei-DNA, which had been autoclaved for 30 min insterile water.

TABLE 5 Effect of autoclaving on MCC and M. phlei-DNA stimulation ofIL-12 production in pg/ml by THP-1 monocytes Non-autoclaved AutoclavedMCC 1 μg/ml 1017.4 905.2 MCC 10 μg/ml 1061.6 1076.8 M. phlei-DNA 1 μg/ml902.0 1088.6 M. phlei-DNA 10 μg/ml 1027.1 949.5

As shown in Table 5, autoclaving does not effect MCC or M. phlei-DNAstimulated IL-12 production by human monocytes.

EXAMPLE 12 Effect of Heat Treatment and of DNase I Treatment on MCC, M.phlei-DNA, MCC-DNA and REGRESSIN® Stimulation of IL-12 Production byMurine Macrophages

Murine peritoneal macrophages were incubated for 48 h with untreatedMCC, M. phlei-DNA, MCC-DNA and REGRESSIN® and with M. phlei-DNA, MCC-DNAand REGRESSIN®, which had been heated at 100° C. for 10 minutes and thencooled in ice for 2 minutes.

TABLE 6 IL-12 production in pg/ml by murine macrophages. IL-12production, pg/ml supernatant Treatment 12.5 μg/ml 5.0 μg/ml 0.1 μg/mlM. phlei-DNA 228 207 140 M. phlei-DNA heat-treated 278 222 164 MCC-DNANot Done 176 164 MCC-DNA heat-treated Not Done 235 131 MCC Not Done 745Not Done REGRESSIN ® 110 96 80 REGRESSIN ® heat-treated 93 82 79

As shown in Table 6, at a concentration of 5 μg/ml, IL-12 production wasstimulated most by MCC, less by M. phlei-DNA and MCC-DNA and least byREGRESSIN®. Heat treatment of M. phlei-DNA, MCC-DNA and REGRESSIN® hadno significant effect on their stimulation of IL-12 production. Althoughnot shown, heat treatment of MCC caused a slight, but significant,increase in IL-12 production.

Murine peritoneal macrophages were incubated for 48 h with untreated andwith DNase I treated MCC, MCC-DNA and REGRESSIN® (FIG. 4). IL-12production was stimulated most by MCC, less by MCC-DNA and least byREGRESSIN®. DNase I treatment of MCC and of MCC-DNA significantlyreduced their stimulation of IL-12 production, whereas DNase I treatmentof REGRESSIN® had no effect on its stimulation of IL-12 production.

These data show that M-DNA, as MCC, M. phlei-DNA or MCC-DNA, stimulatesproduction of the cytokine IL-12 by murine macrophages. As with humanlymphocytes (Example 7), MCC stimulates more IL-12 production than M.phlei-DNA or MCC-DNA and DNase I treatment significantly reduces MCC-DNAand MCC stimulated IL-12 production.

EXAMPLE 13 Effect of Interferon-gamma on MCC Stimulation of IL-12Synthesis by Murine Peritoneal Macrophages

Murine peritoneal macrophages were incubated for 48 h with PBS,interferon-gamma (Life Technologies), MCC and MCC+interferon gamma. Asshown in FIG. 5, in the presence of the PBS, IL-12 production was about75 pg. The addition of 500 units/ml of interferon-gamma had a minimaleffect on IL-12 production. In the presence of 25 μg/ml of MCC, IL-12production increased to about 800 pg. The addition of 500 units/ml ofinterferon-gamma to 25 μg/ml of MCC increased IL-12 production by about180 pg to 980 pg. These results demonstrate that interferon-gammapriming is not required for MCC stimulation of IL-12 production, andthat the effects of interferon-gamma and of MCC are additive. That is,there is no synergy between interferon-gamma and MCC stimulation ofIL-12 production. In contrast, interferon-gamma is a prerequisite forBCG stimulation of IL-12 production.

EXAMPLE 14 Effect of MCC, M-phlei-DNA, MCC-DNA and REGRESSIN® on NitricOxide (NO) Production by Murine Peritoneal Macrophages

Macrophage activation stimulates production of reactive oxygen speciesincluding, but not limited to, nitric oxide, superoxide radicals andhydroxyl radicals. These reactive oxygen species induce cytolysis andapoptosis in responsive cells and, therefore, have anti-cancer activity.

Murine peritoneal macrophages were incubated for 48 h with 0.1, 5.0 or12.5 μg/ml of MCC, M-phlei-DNA, MCC-DNA and REGRESSIN®. NO productionwas measured in nmol/L by reaction of NO₂-with Griess reagent using 100μl of culture supernatant.

TABLE 7 Effect of MCC, M. phlei-DNA, MCC-DNA and REGRESSIN ® on NOproduction by murine macrophages. NO production, nmol/L Treatment 12.5μg/ml 5.0 μg/ml 0.1 μg/ml M. phlei-DNA 18.9 8.3 2.6 MCC-DNA Not Done 3.01.8 MCC Not Done 36.2 Not Done REGRESSIN ® 1.6 2.6 0.1

As shown in Table 7, at 5 μg/ml, MCC, stimulated significantly more NOproduction than M. phlei-DNA or MCC-DNA. REGRESSIN®. stimulated almostno NO production.

Murine macrophages were incubated for 48 h with 1 μg/ml MCC and DNase Itreated MCC, and with MCC-DNA and M. phlei-DNA.

TABLE 8 Stimulation of NO production in murine macrophages by MCC, andDNase I treated MCC and by MCC-DNA and M. phlei-DNA Experiment #1Experiment #2 NO (nmol/ml) NO (nmol/ml) MCC 43.7 30.7 MCC + DNase I (1U) 0.0 2.1 MCC-DNA 2.6 2.1 M. phlei-DNA 0.0 1.6

As shown in Table 8, MCC stimulated significant NO production. DNase Itreatment of MCC abolished MCC stimulation of NO production. MCC-DNA andM. phlei-DNA stimulated minimal NO production.

These data suggest that, for optimal stimulation of NO production bymacrophages, preserved (undegraded) M-DNA must be presented to themacrophages complexed on a carrier such as M. phlei-cell wall.

EXAMPLE 15 Effect of MCC on the Production of Nitric Oxide (NO) byMurine RAW 264.7 Monocytes

Murine RAW 264.7 monocytes were incubated for 24 h with increasingconcentrations of MCC. Increasing concentrations of MCC stimulatedincreasing amounts of NO production (FIG. 6). This was unexpected asreceptors for NO induction are not optimally expressed on monocytes and,therefore, NO production is not usually associated with monocytes. Underthe same conditions, REGRESSIN® did not stimulate NO production.

EXAMPLE 16 Stimulation of Cytokine Synthesis in vivo

Four groups of CD-1 mice, each containing 5 mice, were injectedintraperitoneally with 50 mg/kg of MCC. Blood was collected at 0, 3, 6and 24 h after injection and concentrations (pg/ml) of IL-6, IL-10,IL-12 and GMSF in the sera were determined at 0, 3, 6 and 24 hpost-injection (FIG. 7A). With intraperitoneal MCC, sera concentrationsof IL-6, IL-10 and IL-12 were significantly increased at 3 and 6 hpost-injection, and declined to approximately control values (0 h) at 24h post injection. Sera concentrations of GM-CSF remained at aboutcontrol values (0 h) at 3, 6 and 24 h post-injection.

Five groups of CD-1 mice, each containing 5 mice, were injectedintravenously with 6.6 mg/kg of MCC. Blood was collected at 0, 3, 6 and24 h after injection and concentrations (pg/ml) of IL-10 and IL-12 inthe sera were determined at 0, 3, 6 and 24 h post-injection (FIG. 7B).With intravenous MCC, sera concentrations of IL-12 were significantlyincreased at 3 and 6 h post-injection, and declined to approximatelycontrol values (0 h) at 24 h post injection. Sera concentrations ofIL-10 remained at about control values (0 h) at 3, 6 and 24 hpost-injection.

These data demonstrate that in vivo administration of MCC stimulatesproduction of the anti-cancer cytokine IL-6, IL-10 and IL-12, but notthe pro-cancer cytokine GM-CSF. Further, these data demonstrate that theamount of MCC administered and the route of administration effect theactivity of the MCC.

Four groups of CD-1 mice, each containing 4 mice, were injectedintraperitoneally with untreated and with DNase I-treated MCC, and M.phlei-DNA. After 3 h, the mice were sacrificed, blood was collected bycardiac micropuncture and the concentration (pg/ml) of IL-12 in sera wasmeasured (Table 9).

TABLE 9 Effect of MCC and of M. phlei-DNA ± DNase on IL-12 production inpg/ml by CD-1 mice. MCC + MCC DNase % inhibition mouse #1 255 mouse #5126 49% mouse #2 180 mouse #6  57 68% mouse #3 146 mouse #7 121 17%mouse #4 199 mouse #8 143 28% average 195 ± 46 111 ± 38 40.5 ± 22.6% M.phlei- M. phlei- DNA + DNA DNase % inhibition mouse #9 135 mouse #13 11019% mouse #10 283 mouse #14 146 48% mouse #11 118 mouse #15 121 82%mouse #12 270 mouse #16 169 37% average 195 ± 46 111 ± 38 46.5 ± 26.5%

As shown in Table 9, in vivo administration of MCC and of M. phlei-DNAstimulate production of the anti-cancer cytokine IL-12. After DNase Itreatment, MCC stimulated IL-12 production decreased 40.5% and M.phlei-DNA stimulated IL-12 production decreased 46.5%. This demonstratesthat the oligonucleotides of M-DNA must be preserved for optimalstimulation of IL-12 production in vivo.

EXAMPLE 17 Inhibition of Cell Proliferation

Cell proliferation was determined usingdimethylthiazol-diphenyltetrazolium bromide (MTT) reduction (Mosman etal. Journal of Immunological Methods 65:55-63, 1983). HT-1376, HT-1197,B-16 F1, THP-1, RAW 264.7, Jurkat, HL-60 and HL-60 MX-1 cells wereincubated for 24 h with from 0 μg/ml to 10 μg/ml of MCC (FIG. 8). MCCinhibited proliferation in each of the cancer cell lines tested in adose dependent manner.

HT-1376, HT-1197, B-16 F1, THP-1, RAW 264.7, Jurkat, HL-60 and HL-60MX-1 cells were incubated for 24 h with from 0 μg/ml to 10 μg/ml of M.phlei-DNA, MCC-DNA, herring sperm-DNA and calf thymus-DNA. M. phlei-DNA(FIG. 9A) and MCC-DNA (FIG. 9B) inhibited proliferation in each of thecancer cell lines tested in a dose-dependent manner, whereas herringsperm-DNA (FIGS. 9A & 9B) and calf thymus-DNA (FIGS. 9A & 9B) did notinhibit proliferation of any of the cancer cell lines tested.

Human leukemic THP-1 monocytes were incubated for 24 h with from 0 μg/mlto 10 μg/ml of MCC, M. phlei-DNA, MCC-DNA, and hIL-12. MCC, M. phlei-DNAand MCC-DNA inhibited proliferation of THP-1 monocytes in adose-dependent manner, whereas hIL-12 did not inhibit proliferation ofTHP-1 monocytes (FIG. 10).

HT-1197 and HT-1376 human bladder cancer cells were incubated for 24 hwith from 0 to 100 μg/ml of MCC and LPS. MCC inhibited proliferation ina dose dependent manner in both HT-1197 (FIG. 11A) and HT-1376 (FIG.11B), whereas LPS did not inhibit proliferation in either of the humanbladder cancer cells lines.

As determined by inhibition of cell proliferation, M. phlei-DNA, MCC-DNAand MCC, wherein M-DNA is preserved and complexed on M. phlei cell wall,inhibit proliferation of each of the cancer cell lines tested. Incontrast, LPS, which is a nonspecific immunostimulant reported to induceapoptosis in some cancer cell lines (Izquierdo et al. Anticancer Drugs7:275-2801996); hIL-12, which is a cytokine reported to induce apoptosisin some cancer cell lines (Stine et al. Annals NY Academy of Science795:420-421, 1996), and DNA from calf thymus and from herring sperm donot inhibit proliferation of any of the cancer cell lines tested. Thesedata show that M-DNA inhibits proliferation of the cancer cells testedand that other DNAs (herring sperm-DNA and calf thymus-DNA) cannotreplace M-DNA. These data also show that M-DNA inhibition of cellproliferation does not result from nonspecific immunostimulation (LPS)or from cytokine activity (hIL-12).

EXAMPLE 18 Induction of Apoptosis as Indicated by DNA Fragmentation

Fragmentation of cellular DNA into nucleosome-sized fragments ischaracteristic of cells undergoing apoptosis. Nucleosome-sized fragmentsare DNA fragments possessing a difference of about 200 base-pairs inlength as determined by agarose gel electrophoresis (Newell et al.Nature 357:286-289, 1990). To assess DNA fragmentation, non-adherentcells were collected by centrifugation at 200 g for 10 min. Pellets ofnon-adherent cells and the remaining adherent cells were lysed with 0.5ml of hypotonic lysing buffer (10 mM Tris buffer, 1 mM EDTA, 0.2% TritonX-100, pH 7.5). The lysates were centrifuged at 13,000 g for 10 min andthe supernatants, containing fragmented DNA, were precipitated overnightat −20° C. in 50% isopropanol and 0.5 M NaCl. The precipitates werecollected by centrifugation and were analyzed by electrophoresis in 0.7%agarose gels for 3 h at 100V.

A suspension culture of human leukemic THP-1 monocytes was incubated for48 h with PBS and with 1 μg/ml of untreated and DNase I treated MCC andM. phlei-DNA and with herring-sperm DNA (FIG. 12). MCC (lane 4) and M.phlei-DNA (lane 2) induced significant DNA fragmentation, whereas PBS(lane 1), DNase I treated MCC (lane 5), DNase I treated M phlei-DNA(lane 3), and herring sperm-DNA (lane 6) did not induce significant DNAfragmentation. A 123-bp DNA ladder (Life Technologies) was used todetermine the molecular weight of the nucleosome-sized DNA fragments(lane L).

HT-1197 and HT-1376 human bladder cancer cells were incubated for 48 hwith 1 μg/ml MCC or hIL-12. MCC induced DNA fragmentation innon-adherent HT-1197 cells, but not in adherent HT-1197 cells (FIG. 13A)MCC also induced DNA fragmentation in non-adherent HT-1376 cells, butnot in adherent HT-11376 cells (FIG. 13B). hIL-12 did not induce DNAfragmentation in HT-1197 (FIG. 13A) or HT-1376 cells (FIG. 13B). A123-bp DNA ladder (Life Technologies) was used to determine themolecular weight of the nucleosome-sized DNA fragments (lane L).

As determined by nuclear fragmentation, M. phlei-DNA and MCC, whereinM-DNA is preserved and complexed on M. phlei cell wall, induce apoptosisin human leukemic THP-1 monocytes and in HT-1 197 and HT-1376 humanbladder cancer cells, whereas herring sperm-DNA, hIL-12, and DNase Itreated M. phlei DNA and MCC do not induce apoptosis in these cells.These data show that the that the oligonucleotides of M-DNA must beintact (DNase I treatment) for induction of apoptosis and that otherDNAs (herring sperm-DNA) cannot replace M-DNA. These data also show thatM-DNA induction of apoptosis does not result from nonspecificimmunostimulation (LPS).

EXAMPLE 19 Induction of Apoptosis as Indicated by Solubilization ofNuclear Mitotic Protein Apparatus (NuMA)

Striking morphological changes in the cell nucleus caused by thesolubilization and release of NuMA are characteristic of apoptosis. Todetermine solubilization and release of NuMA, media from the culturedcells were removed and centrifuged at 200 g for 10 min. The supernatantswere collected and 100 μl of each supernatant were used to quantitateNuMA release in units/ml (U/ml) using a commercial ELISA (Calbiochem,Cambridge, Mass.) (Miller et al. Biotechniques 15:1042-1047, 1993).

Human leukemic THP-1 monocytes were incubated for 48 h with 0 μg/ml to10 μg/ml of MCC, M. phlei-DNA, MCC-DNA, and herring sperm-DNA. MCC, M.phlei-DNA and MCC-DNA induced release of NuMA from THP-1 monocytes in adose-dependent manner, whereas herring sperm-DNA did not induce releaseof NuMA (FIG. 14). MCC was more effective than M. phlei-DNA or MCC-DNAin inducing apoptosis in THP-1 monocytes. DNase I treatment of MCC, M.phlei-DNA and MCC-DNA significantly inhibited their induction of NuMArelease from these cells (FIG. 15).

HT-1197 and HT-1376 human bladder cancer cells were incubated with 0μg/ml to 100 μg/ml of MCC. MCC induced the release of NuMA in adose-dependent manner (FIG. 16). HT-1197 and HT-1376 human bladdercancer cells were incubated with 1 μg/ml and with 100 μg/ml of MCC. MCCinduced release of NuMA in a time-dependent manner (FIGS. 17A & 17B).Enhanced release of NuMA was detected within 24 hours after incubationof HT-1197 cells (FIG. 17A) and of HT-1376 (FIG. 17B) cells with 100μg/ml of MCC.

As determined by NuMA release, MCC, M. phlei-DNA and MCC-DNA, each ofwhich contain M-DNA, induce apoptosis in the cancer cell lines tested.MCC, wherein M-DNA is preserved and complexed on M. phlei cell wall,induces more apoptosis than M. phlei-DNA or MCC-DNA. This suggests thatthe carrier that presents M-DNA to responsive cells effects M-DNAinduction of apoptosis.

EXAMPLE 20 Fas Independent Induction of Apoptosis in Jurkat Human TLymphoblast Cells by M. phlei-DNA

Jurkat human T lymphoblast cells were incubated for 1 h with PBS, withCH-11 antibody (1 μg/ml), an antibody that binds to Fas and inducesapoptosis (+control) or with ZB4 antibody (1 μg/ml), an antibody thatbind to Fas and inhibits apoptosis (−control) (Coulter-Immunotech). M.phlei-DNA (1 μg/ml) was added and NuMA release was determined after 48h.

As shown in FIG. 18, M. phlei-DNA induced apoptosis both alone and inthe presence of ZB4antibody. These data demonstrate that M. phlei DNAinduction of apoptosis is independent of Fas.

EXAMPLE 21 Summary of Effects of MCC, M phlei-DNA and MCC-DNA on CellProliferation and on Apoptosis

Table 10 summarizes the effects of MCC, M phlei-DNA and MCC-DNA on cellproliferation and on apoptosis induction as determined by DNAfragmentation, NuMA release, and flow cytometric analysis in a varietyof human and murine cancer cell lines.

TABLE 10 Inhibition of proliferation and induction of apoptosis in humanand murine cancer cell lines Flow Inhibition of Nucleosome- NuMAcytometric Cells proliferation sized DNA released analysis THP-1 yes yesyes  ND* HL-60 yes yes yes ND HL-60 MX-1 yes yes yes ND RAW 264.7 yesyes yes ND JURKAT yes yes yes ND HT-1376 yes yes yes ND HT-1197 yes yesyes ND B-16-FI yes yes ND ND SW260 ND ND ND yes OC2 ND ND ND yes LS1034yes ND yes ND *ND = Not Done

MCC, M. Phlei-DNA and MCC-DNA inhibit proliferation and induce apoptosisin each of the cancer cell lines tested. These cancer cell lines includeatypical drug resistant HL-60 MX-1 human promyelocytic leukemia, p53/p21abnormal and drug resistant HT-1376 human bladder, Fas abnormal SW260human colon, and conventional drug resistant LS1034 human cecumcarcinoma cells.

EXAMPLE 22 MCC Activation of Caspase-3 in Human Leukemic THP-1 Monocytes

Caspase 3 is a key enzyme in the apoptotic pathway downstream ofFas-FasL signaling. To determine if MCC can by-pass Fas and directlyactivate the caspase cascade in cancer cells, the effect of MCC oncaspase-3 activity was assayed in human leukemic THP-1 monocytes.

THP-1 monocytes (2×10⁷ cells) were incubated for 48 h with MCC (100μg/ml). The THP-1 cells were lysed in 50 mM HEPES, pH 7.4, 100 mM NaCl,0.1% (3-[3-cholamidopropyl)-dimethyl-ammonio]-1-propane-sulfonate,CHAPS, 10 mM DTT, 1 mM EDTA and 10% glycerol. Caspase-3 activity wasdetermined with a commercial ELISA (BIOMOL Research Laboratories, Inc.,Plymouth Meeting, Pa.), using the included substrate, inhibitor andpurified caspase-3 enzyme. Results are expressed as optical densitiesread at 405 nm.

TABLE 11 MCC (100 μg/ml) activation of caspase 3 activity in humanleukemic THP-1 monocytes. p-nitroanalide absorbance, 405 nm O.D. × 10⁻¹,O.D. × 10⁻¹, Incubation 3 hours incubation 6 hours incubation THP-1alone 0.19 0.06 THP-1 + MCC cell extract 0.44 0.20 THP-1 + MCC treatedwith 0.12 0.11 DNase cell extract Purified Caspase-3 0.87 0.58 PurifiedCaspase-3 + 0.00 0.00 caspase-3 inhibitor THP-1 + MCC cell extract +0.00 0.00 Caspase-3 inhibitor

As shown in Table 11, incubation with MCC induced a 232% (3 h) and a333% (6 h) increase in caspase-3 and caspase-3 like activity in humanleukemic THP-1 monocytes. MCC induction of caspase-3 and caspase-3 likeactivity was abolished by DNase I treatment of MCC. Specificity of MCCinduction of caspase-3 and caspase-3 like activity was demonstratedusing caspase-3 inhibitor. Addition of caspase-3 inhibitor toMCC-treated THP-1 cell extracts completely abolished measurableactivity. The ability of MCC to directly and specifically inducecaspase-3 and caspase-3 like activity in human leukemic THP-1 monocytesis totally unexpected. To induce caspase-3 and caspase-3 like activity,MCC must be gain entry into the cells by one or more mechanisms and,then, directly induce caspase activity to initiate the lethalproteolytic cascade of apoptosis execution.

EXAMPLE 23 Effect of Tamoxifen on MCC Inducted Apoptosis in HumanLeukemic THP-1 Monocytes

Human leukemic THP-1 monocytes were incubated for 90 min in controlmedium or in medium containing 10 μg/ml((Z)-2-[p-(1,2-Diphenyl-1-butenyl)-phenoxy]-N,N-dimethylethylamine),tamoxifen, (Sigma-Aldrich), an anti-estrogen used in the palliativetreatment of advanced breast cancer. Cells were washed extensively withice-cold medium (2×), resuspended to about 10⁶ cells/ml in medium andincubated for 48 h with 0, 1, 10 and 100 μg/ml of MCC. Apoptosis wasquantitated by measuring NuMA.

TABLE 12 Effect of tamoxifen on MCC induced apoptosis in human leukemicTHP-1 monocytes determined by NuMA release in U/ml. +MCC +MCC +MCC+medium 1 μg/ml 10 μg/ml 100 μg/ml only Control 174.6 260.0 237.2 174.6Tamoxifen 354.9 406.2 410.0 284.7 (10 μg/ml) (↑ 50.8%) (↑ 36.0%) (↑42.1%)

As shown in Table 12, preincubation in tamoxifen significantly increasedMCC induced apoptosis at each of the MCC concentrations used. These datademonstrate that MCC can be used as an adjunct to other anti-canceragents to increase treatment effectiveness.

EXAMPLE 24 Modification of M-phlei-DNA by Methylation, Sonication andAutoclaving

The activity of BCG nucleic acid is abolished completely by cytosinemethylation of BCG with CpG methylase (Krieg et al. Nature 374:546-549,1995). Therefore, the effect of CpG methylation on the ability of M.phlei-DNA to induce apoptosis was determined.

M. phlei-DNA, 1 μg, was methylated using 2.5 U of CpG Sss I methylase(New England Biolabs, Mississauga, Ontario, Canada) for 1 h at 37° C.Native and methylated M. phlei-DNA were subjected to cleavage by BstU Irestriction endonuclease (New England Biolabs) for 1 h at 60° C. andwere analyzed by electrophoresis in 0.5% agarose gel (3 h, 100 V).

As shown in FIG. 19, untreated M. phlei-DNA was digested by BstU Irestriction endonuclease (lanes 1 and 2), whereas methylated M.phlei-DNA (lanes 3 and 4) was not digested by BstU I restrictionendonuclease. This confirms that methylation of the M. phlei-DNA wascomplete. Untreated M. phlei-DNA was included as a control (lanes 5 and6). The 123 bp DNA ladder (Life Technologies) was used to determine themolecular size of digested DNA fragments (lane L).

As shown in FIG. 20, methylation did not modify M. phlei-DNA inducedrelease of NuMA from human leukemic THP-1 monocytes. Therefore, unlikeBCG-DNA, CG-motifs are not necessary for apoptosis induction by M.phlei-DNA.

The oligonucleotide length of M phlei-DNA (1 μg) was reduced bysonication for 15 sec or for 20 min on ice in a Model W-38 ultrasonicprocessor (HeatSystems-Ultrasonics, Inc.), by autoclaving at 121° C. for30 min (Castle Sybron MDT, Dubuque, Iowa), or by digestion with BstU Irestriction endonuclease. Sonication, autoclaving and BstU I digestion,each of which reduce oligonucleotide length to the range of about 5 basepairs to about 250 base pairs, did not change M. phlei-DNA induction ofNuMA release from THP-1 monocytes (FIG. 20). Similar results wereobtained with MCC. These results demonstrate that M. phlei-DNA and MCCinduce apoptosis in cancer cells, even at short base-pair length (about5 base pairs to about 250 base pairs).

Human leukemic THP-1 monocytes were incubated for 48 h with untreatedMCC and M. phlei-DNA and with MCC and M. phlei-DNA autoclaved for 30 minat 121° C. Autoclaving did not affect the ability of MCC or of M.phlei-DNA to inhibit of proliferation of (Table 13A), or to induceapoptosis in (Table 13B) human leukemic THP-1 monocytes.

TABLE 13A Effect of autoclaving on MCC and M. phlei-DNA inhibition ofproliferation Non-autoclaved Autoclaved % inhibition % inhibition MCC 1μg/ml 87 ± 4  84 ± 10 MCC 10 μg/ml 68 ± 1 74 ± 6 MCC 100 μg/ml 59 ± 7 64± 6 M. phlei-DNA * 1 μg/ml 88 ± 8  84 ± 11 M. phlei-DNA 10 μg/ml 80 ± 874 ± 6 M. phlei-DNA 100 μg/ml 68 ± 4 64 ± 5

TABLE 13B Effect of autoclaving on MCC and M. phlei-DNA induction ofapoptosis determined by NuMA release in U/ml. Non-autoclaved AutoclavedMCC 1 μg/ml 298.0 277.1 MCC 10 μg/ml 325.9 322.4 MCC 100 μg/ml 339.9357.3 M. phlei-DNA * 100 μg/ml 261.4 268.4 M. phlei-DNA 10 μg/ml 317.2278.4 M. phlei-DNA 1 μg/ml 306.8 285.8

EXAMPLE 25 MCC Inhibits Cancer Growth in vivo

MCC and DNase I treated MCC were emulsified to a final concentration of1 mg/ml in PBS containing 2% w/v mineral oil and 0.02% w/v TWEEN 80(Fisher Chemical Co.) by sonication at 4° C. for 5 min.

Line 10 hepatoma cells, syngenic for strain-2-guinea pigs were rapidlythawed, washed by centrifugation and resuspended in M199 medium to aconcentration of 10⁶ cells/ml. One-tenth ml containing 1.5×10⁶ cells wasinjected intradermally into the flanks of 3 month-old strain 2 guineapigs. Treatment was initiated 6 to 7 days post-injection, when thecancers were between about 0.5 and about 0.8 cm in diameter. Sevenanimals were treated with emulsification buffer alone (control), 7 withemulsification buffer containing MCC, and 7 with emulsification buffercontaining DNase I treated MCC. The emulsions were instilled directlyinto the cancer and surrounding normal tissue. One-half ml of emulsionwas administered at 0 h and at 6 h for a total volume of 1 ml containing1 mg of MCC or of DNase I treated MCC.

Cancer diameters (longest diameter+shortest diameter) were recordedweekly for 3 weeks. Cancer volumes were calculated in mm³ as 0.5×a(longest diameter)×b² (shortest diameter) and the increase in cancervolume relative to day 0 of treatment was calculated for each guineapig. Statistical analysis was done using 2-way ANOVA with replicates(PHARM/PCS version 4.2, MCS, Philadelphia, Pa.). Differences intreatment were considered significant at p≦0.05.

As shown in FIG. 21, with control emulsion, cancer volume increased byabout 22-fold by week 3, whereas with MCC, cancer growth wassignificantly inhibited compared to control emulsion (FIG. 21, Table14). With DNase I treated MCC, cancer growth was not significantlydifferent from control (FIG. 21, Table 14).

TABLE 14 Two-way ANOVA with replicates Treatment comparison p value Control vs MCC p < 0.01 Control vs MCC + DNase Not significant MCC vsMCC + DNase p < 0.01

These data show that instillation of MCC at the site of a tumor resultsin regression of the tumor. Moreover, the significant (p<0.01)difference in inhibition of cancer growth between MCC and DNase Itreated MCC demonstrates that the oligonucleotides of M-DNA must bepreserved (undegraded) for the anti-cancer activity of MCC in vivo.

EXAMPLE 26 MCC Cytotoxicity

Cell cytotoxicity is characterized by the loss of plasma membraneintegrity and release of cytoplasmic enzymes such as, but not limitedto, LDH (Wyllie et al. International Review of Cytology 68: 251-306,1980; Phillips et al. Vaccine, 14:898-904, 1996). Human bladder cancercells release LDH when treated with cytotoxic agents (Rahman M. UrologyInternational 53:12-17, 1994).

To assess the cytotoxicity of MCC, HT-1197 and HT-1376 human bladdercancer cells were incubated for 48 h with from 0 μg/ml to 100 μg/ml ofMCC or with lysing buffer (10 mM Tris, 1 mM EDTA, 0.2% Triton X-100, pH7.5) as a control for total LDH release (Filion et al. Biochim BiophysActa 1329:345-356, 1997). LDH release into the culture supernatant wasdetermined using a commercial assay (Sigma-Aldrich).

As determined by LDH release, MCC was not cytotoxic to HT-1197 or toHT-1376 cells (FIG. 22). The absence of cytotoxicity demonstrates thatMCC acts directly to inhibit proliferation of and to induce apoptosis incancer cells.

EXAMPLE 27 MCC Stability

MCC at 1 mg/ml was stored as a sterile suspension in 0.85% w/v NaCl inthe dark at 4° C. or 6 months. Mean particle diameter was calculatedusing photon correlation spectroscopy (N4 Plus, Coulter ElectronicsInc.). The MCC suspension was diluted with 0.85% w/v NaCl to a particlecount rate between 5×10⁴ and 10⁶ counts/sec. Mean particle diameter wascalculated in size distribution processor mode (SDP) using the followingconditions: fluid refractive index 1.33, temperature 20° C., viscosity0.93 centipoise, angle of measurement 90.0°, sample time 10.5 μs, andsample run time 100 sec. Potential, the electric charge at thehydrodynamic interface between the particles and the bulk solvent, wasmeasured in a Delsa 440SX (Coulter Electronics Inc.) using the followingconditions: current 0.7 mA, frequency range 500 Hz, temperature 20° C.,fluid refractive index 1.33, viscosity 0.93 centipoise, dielectricconstant 78.3, conductivity 16.7 ms/cm, on time 2.5 sec, off time 0.5sec, and sample run time 60 sec.

As shown in FIG. 23, MCC charge and MCC diameter remained relativelyunchanged during 6 months of storage. Moreover, MCC stimulation of IL-12production in THP-1 monocytes and MCC induction of apoptosis in THP-1monocytes remained unchanged during 6 months of storage.

EXAMPLE 28 MCC and MCC-DNA Treatment of Human Colon Cancer

Human colon cancer cells (ICM12C) are established as an ectopic solidtumor in the subcutaneous tissues of immunodeficient athymic nude mice(nu/nu mice) and the mice are divided into 5 groups. Group 1 receivesvehicle alone. Group 2 receives MCC. Group 3 receives DNase I treatedMCC. Group 4 receives MCC-DNA. Group 5 receives DNase I treated MCC-DNA.Cancer mass is measured before treatment and weekly during 4 weeks oftreatment. Group 2 mice and Group 4 mice show regressin of cancer mass.

EXAMPLE 29 MCC and M. phlei-DNA Treatment of Human Ovarian Cancer

Human ovarian cancer cells (36M2) are established as ascites in theperitoneal cavity of immunodeficient athymic nude mice (nu/nu mice) andthe mice are divided into 5 groups. Group 1 receives vehicle alone.Group 2 receives MCC. Group 3 receives DNase I treated MCC. Group 4receives M. phlei-DNA. Group 5 receives DNase I treated M. phlei-DNA.Cancer mass is measured before treatment and weekly during 4 weeks oftreatment. Group 2 mice and Group 4 mice show inhibition of asciticcancer cell proliferation.

EXAMPLE 30 MCC Treatment of Canine Transmissible Venereal Cancer

Dogs with veneral cancers (VT) are divided into 4 groups. Group 1receives vincristine. Group 2 receives vincristine combined withmethotrexate and cyclophosphamide. Group 3 receives MCC. Group 4receives vincristine and MCC. Cancer mass is measured before treatmentand weekly during 12 weeks of treatment. Group 3 and Group 4 dogs showregression of VT. Group 4 dogs show more regression of VT than Group 3dogs.

EXAMPLE 31 MCC Treatment of Canine Osteosarcoma

Dogs with osteosarcoma are treated by amputation followed by MCC and MCCplus cisplatin weekly for 4 weeks. They are then treated weekly for 4additional weeks with MCC plus cisplatin. Both the MCC and theMCC-cisplatin are effective.

EXAMPLE 32 Suspension in Aqueous Buffer

Lyophilized MCC is suspended in a pharmaceutically acceptable buffer andis emulsified by sonication at 20% output for 5 minutes (Model W-385Sonicator; Heat Systems-Ultrasonics Inc). Optionally, the emulsifiedmixture is homogenized by microfluidization at 15,000-30,000 psi for oneflow-through (Model M-110Y; Microfluidics, Newton, Mass.). Thesuspension is either processed under aseptic conditions or is sterilizedby autoclaving.

EXAMPLE 33 Emulsification of MCC in Neutral Lipid.

DNase free phosphatidylcholine is added to DNase free triglyceridesoybean oil at a ratio of 1 gram of phospholipid to 20 ml oftriglyceride and is dissolved by gentle heating at 50°-60° C. Severalgrams of MCC are added to a dry autoclaved container and thephospholipid-triglyceride solution is added at a concentration of 20 mlper 1 gram of MCC. The suspension is incubated for 60 min. at 20° C. andis then mixed with DNase-free PBS in the ratio of 20 ml MCC suspensionper liter of PBS. The mixture is emulsified by sonication at 20% outputfor 5 minutes (Model W−385 Sonicator; Heat Systems-Ultrasonics Inc.).Optionally, the emulsified MCC mixture is homogenized bymicrofluidization at 15,000-30,000 psi for one flow-through (ModelM-110Y; Microfluidics). The MCC emulsion is transferred to anautoclaved, capped bottle for storage at 4° C.

It should be understood, of course, that the foregoing relates only to apreferred embodiment of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

1 1 15 DNA Unknown Description of Artificial Sequence synthetic 1gctagacgtt agcgt 15

We claim:
 1. A composition, comprising: a. Mycobacterium phlei (M.phlei) deoxyribonucleic acid (M-DNA); b. deproteinized, delipidated M.phlei cell wall, wherein the M-DNA is preserved and complexed on the M.phlei cell wall (MCC); and c. a pharmaceutically acceptable carrier. 2.The composition of claim 1, wherein the pharmaceutically acceptablecarrier is selected from the group consisting of a liquid carrier and asolid carrier.
 3. A method for inhibiting the growth of cancer cells ina mammal having cancer, wherein a composition comprising: a.Mycobacterium phlei (M. phlei) deoxyribonucleic acid (M-DNA); b.deproteinized, delipidated M. phlei cell wall, wherein the M-DNA ispreserved and complexed on the M. phlei cell wall (MCC); and c. apharmaceutically acceptable carrier is administered to the mammal havingthe cancer in an amount effective to inhibit the growth of the cancercells in the mammal having the cancer.
 4. The method of claim 3, whereinthe pharmaceutically acceptable carrier is selected from the groupconsisting of a liquid carrier and a solid carrier.
 5. The method ofclaim 3, wherein the growth of the cancer cells is inhibited byinduction of apoptosis in the cancer cells.
 6. The method of claim 5,wherein the induction of apoptosis is independent of abnormal Fas. 7.The method of claim 5, wherein the induction of apoptosis is independentof abnormal p53/p21.
 8. The method of claim 5, wherein the induction ofapoptosis is independent of drug resistance.
 9. The method of claim 3,wherein the growth of the cancer cells is inhibited by stimulation ofimmune system cells to produce bioactive molecules.
 10. The method ofclaim 9, wherein the bioactive molecules are selected from the groupconsisting of cytokines and reactive oxygen species.
 11. The method ofclaim 10, wherein the cytokines are selected from the group consistingof IL-6, IL-10 and IL-12.
 12. The method of claim 11, wherein thecytokine is IL-12.
 13. The method of claim 3, wherein the cancer isselected from the group consisting of leukemia, lymphoma, melanoma,bladder cancer, colon cancer, esophageal cancer and cecal cancer. 14.The method of claim 13, wherein the cancer is bladder cancer.
 15. Themethod of claim 3, wherein the growth of the cancer cells is inhibitedby inhibition of proliferation of the cancer cells.
 16. The method ofclaim 3, wherein the growth of the cancer cells is inhibited byactivation of caspases in the cancer cells.
 17. A method for inhibitingthe growth of cancer cells in a mammal having cancer, wherein acomposition comprising: a. Mycobacterium phlei (M. phlei)deoxyribonucleic acid (M-DNA) and b. a DNase free pharmaceuticallyacceptable carrier is administered to the mammal having the cancer in anamount effective to inhibit the growth of the cancer cells in the mammalhaving the cancer.
 18. The method of claim 17, wherein thepharmaceutically acceptable carrier is selected from the groupconsisting of a liquid carrier and a solid carrier.
 19. The method ofclaim 17, wherein the growth of the cancer cells is inhibited byinduction of apoptosis in the cancer cells.
 20. The method of claim 19,wherein the induction of apoptosis is independent of abnormal Fas. 21.The method of claim 19, wherein the induction of apoptosis isindependent of abnormal p53/p21.
 22. The method of claim 19, wherein theinduction of apoptosis is independent of drug resistance.
 23. The methodof claim 17, wherein the growth of the cancer cells is inhibited bystimulation of immune system cells to produce bioactive molecules. 24.The method of claim 23, wherein the bioactive molecules are selectedfrom the group consisting of cytokines and reactive oxygen species. 25.The method of claim 24, wherein the cytokines are selected from thegroup consisting of IL-6, IL-10 and IL-12.
 26. The method of claim 25,wherein the cytokine is IL-12.
 27. The method of claim 17, wherein thecancer is selected from the group consisting of leukemia, lymphoma,melanoma, bladder cancer, colon cancer, esophageal cancer and cecalcancer.
 28. The method of claim 27, wherein the cancer is bladdercancer.
 29. The method of claim 17, wherein the growth of the cancercells is inhibited by inhibition of proliferation of the cancer cells.30. The method of claim 17, wherein the growth of the cancer cells isinhibited by activation of caspases in the cancer cells.